Transplantation of isolated parenchymal cells has great promise as a minimally invasive therapy and partial success has been reported for several rare metabolic liver diseases. Major limitations to the technique include an inability to transplant an adequate mass of donor hepatocytes, and lack of a sensitive enough technology to monitor the status of transplanted cells. This proposal includes several major innovations in understanding hepatocyte transplant biology and optimizing its application. In animal models liver-directed radiation facilitates repopulation of the native liver by transplanted hepatocytes. Unfortunately, the standard clinical and laboratory signs that identify rejection in solid organ transplantation are not useful in recipients of cell transplants, and the sensitivity of functional changes following hepatocyte transplant is inadequate to diagnose rejection before damage to the allograft is irreversible. The availability of assays that would allow non-invasive monitoring of graft function and assessment of rejection risk would facilitate more rational management of patients following hepatocyte transplantation. As diseases targeted for treatment by hepatocyte transplantation are rare, and most reports involve anecdotal experience in a diverse patient population, we will transplant patients with phenylketonuria (PKU), a disease with an incidence that will make adequate patient enrollment possible. Phenylalanine hydroxylase (PAH) deficiency, traditionally known as PKU was the original motivation for population-based newborn screening. However, standard dietary management strategies have failed in older adolescents and adults due to difficulty in adhering to diet, leading to diminished executive function, and other neurologic and neuropsychiatric problems. The proposal has three specific aims. Aim 1 is to confirm the safety of liver-directed radiation conditioning and document 5-10% replacement of host hepatocytes by donor hepatocytes. We hypothesize that use of multiple donors and pre-transplant hepatic radiation conditioning will lead to competitive repopulation of the host liver and clinical correction of PAH deficiency in patients with PKU, a model disease for treatment of liver-based metabolic diseases. Aim 2 is to identify graft rejection by immunologic monitoring and to successfully treat it before the process is irreversible. We hypothesize that monitoring rejection risk by a proven assay of donor-specific T cell immune reactivity can be used to assess the adequacy of immune suppression following cell transplantation. Aim 3 is to determine the extent to which use of real-time measures of donor hepatocyte function, rather than use of surrogates, will allow assessment of graft function after hepatocyte transplantation. We hypothesize that isotopic Phe turnover studies in PKU patients can more effectively measure changes in Phe metabolism than standard measures. These results will be important for the possible transplantation of gene edited autologous or stem cell-derived hepatocytes. Delivery of gene editing vectors ex vivo to hepatocytes is significantly more efficient than in vivo delivery, and is even more efficient in patient-derived fibroblasts that would later be converted to iPS-derived hepatocytes.